The primary humoral immunodeficiency, X-linked agammaglobulinemia (XLA), results from deficient function of Bruton's tyrosine kinase (Btk). This disorder is characterized by a block in early B-cell development leading to a profound reduction in the generation, activation, and survival of mature B lineage cells. Affected patients exhibit a marked reduction in circulating immunoglobulin levels, and recurrent bacterial infections. A range of compelling observations documents the strong selective pressure for B lineage cells expressing a normal Btk gene. These observations suggest that introduction of a the wild type Btk cDNA in autologous HSC will lead to long-term immunologic reconstitution in XLA patients. This approach, if successful, would be predicted to lead to improved quality of life and increased life expectancy in patients with XLA. Our laboratory has carried out extensive studies of onco-retroviral based Btk gene therapy in Btk deficient models including: 1.) Transplantation of transduced of IL-7 expanded, Btk deficient murine bone marrow B progenitors into SCID mice; and 2.) Transplantation of transduced Btk deficient HSC into SCID or Btk deficient recipients. However, despite use of retroviral vectors optimized for expression in HSC populations; efficient and sustained cell marking; sustained B lineage engraftment and selective expansion of marked B lineage cells; this approach has lead to only partial reconstitution of Btk dependent B cell development and function. Most notably, this approach has failed to rescue Btk dependent, B cell receptor (BCR) signaling. Rescue of BCR signaling is critical for restoration of normal humoral function in XLA. Notably, the incomplete response observed in these studies correlates with sub-endogenous levels of Btk expression (below 50%) in marked B lineage cells in vivo. Our data suggest that this results from suboptimal LTR dependent transcription in the mature B lineage pool. In order to overcome the problems associated with onco-retroviral Btk gene transfer, we have initiated a series of alternative approaches for restoration of Btk function in B lineage populations. We will test the hypotheses that: 1) An optimized lentiviral vector containing a highly active, B-lineage specific promoter/enhancer will permit long-term high level Btk gene expression and rescue of B cell function; 2) Btk expression vectors containing a cis-linked cell expansion signal (triggered by an in vivo chemical inducer of dimerization) will permit expansion of transduced B lineage progenitors and further enhance the selective outgrowth of the highest Btk expressing progenitors; and 3.) The combination of direct genomic modification of Btk deficient stem cells (using RNA/DNA molecules), and the strong selective advantage for gene corrected progenitors, will rescue of Btk deficient function in a Btk missense mutant mouse model.